It is known that a foreign DNA will be integrated into chromosome via repairing mechanism at the time of double-strand break (DSB) of chromosomal DNA. There are two kinds of mechanisms in the repairing, that is, homologous recombination and non-homologous recombination (non-homologous end joining). The integration will occur through a region having homology with the foreign DNA in the case of the homologous recombination. On the other hand, the integration will do at a random site of the chromosome regardless of a sequence of the foreign DNA in the case of the non-homologous recombination. It is conceived that the two recombination mechanisms will function in equilibration (Ristic et al., Nucl. Acids Res. (2003) 31:5229-5237).
A series of genes belonging to a so-called “rad52 group” take an essential role in the homologous recombination, which includes rad50, 51, 52, 54, Mrell and XRS2 (Kooistra et al. 2004). The homologous recombination mechanism has been confirmed to exist in a wide range of organisms from bacteria to eucaryotic organisms. A uvsC gene has been cloned and studied using Aspergillus nidulans, an experimental strain belonging to Aspergillus, having a mononuclear conidium (van Heemst et al., Mol. Gen. Genet (1997) 254:654-64), and it was reported that the frequency of the homologous recombination would be improved by increasing expression frequency of the above genes up to a certain level.
On the other hand, it has been revealed that the non-homologous recombination will proceed with non-homologous end joining mechanism that is completely different form the homologous recombination mechanism. Genes such as ku70, ku80, Xrcc4, LIG4 and DNAPKcs are known to take an essential role in this recombination mechanism. It is known that Ku70 and Ku80 will act as a hetero dimmer, form a complex with a nucleotide kinase (XRCC4) and DNA Ligase IV, and promote the non-homologous end joining by joining with a DNA end at the time of cleavage of the DNA double-strand break for its repairing (Walker et al., Nature (2001) 412:607-614). The non-homologous recombination via ku gene has been recognized only in eucaryotic organisms.
Changes in phenotype due to the mutation or disruption of the ku gene have been reported in yeast, animal cells, plant cells, etc. The disruption of the ku gene would cause temperature-sensitiveness in Saccharomyces cerevisiae (Silmon et al., 1996: Non-Patent Document 1), aplasia and smallness in mice (Nussenzweig et al., 1996: Non-Patent Document 2), and binding to telomere ends and involvement in keeping their stability in human (Hsu et al 2000: Non-Patent Document 3). Furthermore, it is known that the disruption of the ku gene would increase telomere length and sensitivity against MMS in plant (Bundock et al., 2002: Non-Patent Document 4). It is also known that the ku gene would not affect non-homologous recombination via T-DNA (Gallego et al., 2003: Non-Patent Document 5). It is then deduced from the above documents that it is virtually impossible to predict the results of disruption of the ku gene because it can cause various changes in phenotypes.
Recently, it was reported that mutation in the ku gene would increase the targeting frequency in yeast of Kluyveromayces lactis (Kooistra et al., 2004: Non-Patent Document 6) and in Neurospora crassa (Ninomiya et al., 2004. Non-Patent Document 7). However, the yeast in Non-Patent Document 6 originally could show such a high targeting frequency as 88% when a homologous region had a length of about 600 bp, and its increase percentage was at most ten and several %. Similarly, the Neurospora crassa in Non-Patent Document 7 originally could show such a high targeting frequency as about 20% when a homologous region had a length of about 1 kb, and the frequency was increased at most by 5 times. The Neurospora crassa belongs to multinuclear fungi having sexual generation.
Japanese Patent Publication 2003-526376 (Patent Document 1) discloses a method for improving homologous recombination, by means of, for example, a ligation inhibitor at non-homologous ends such as anti-Ku antibody and Ku antisense RNA, or a homologous recombination accelerator such as Rad52 protein. However, it does not contain any actual examples, especially it does not have any disclosure or suggestion with respect to an example using filamentous fungi such as Asperigillus. 
On the contrary, the gene targeting frequency in the case of Aspergillus was as very low as 1˜3% even if a homologous region has about 2 kb. Accordingly, it has been recognized that it would require a lot of efforts to obtain a desired gene-disruption strain in case a screening on the basis of phenotypes was impossible (Takahashi et al., 2004: Non-Patent Document 8).    [Patent Document 1] Japanese Patent Publication 2003-526376    [Non-Patent Document 1] Silmon et al., Nucl. Acds Res. (1996) 24:4639-4684    [Non-Patent Document 2] Nussenzweig et al., Nature (1996) 382: 551-555    [Non-Patent Document 3] Hsu et al., Genes & Development (2000) 14: 2807-2812    [Non-Patent Document 4] Bundock et al., Nucl. Acids Res. (2002) 30:3395-3400    [Non-Patent Document 5] Gallego et al., Plant J (2003) 35:557-565    [Non-Patent Document 6] Kooistra et al. Yeast (2004) 21: 781-792    [Non-Patent Document 7] Ninomiya et al., PNAS (2004) 101:12248-12252    [Non-Patent Document 8] Takahashi et al., Mol. Gen. Genet. (2004) 272:344-52
Aspergillus strains such as Aspergillus sojae and Aspergillus oryzae are industrially used in the production of brewed food such as soy sauce, sake (rice wine), soybean paste, etc. Recently, a genomic sequence of Aspergillus oryzae has been identified, and functional analysis of their genes has become more important.
However, unlike Aspergillus nidulans, niger, fumigatus and awamori that have a mononuclear generation, Aspergillus sojae and Aspergillus oryzae are always kept in a multinuclear state in their whole life cycle including in a conidium condition, and their sexual generation has not yet been observed. Their nuclear-distribution mechanism from a parent cell to a daughter cell has not yet been revealed, either. Accordingly, a mutant cannot be produced by means of mating between strains or RIP (Repeat Induced Mutation), which makes it difficult to study their genetics. As a result, the genetic analysis of Aspergillus sojae and Aspergillus oryzae has fallen behind in spite of their industrially very high utility.
Gene disruption or gene-replacement by gene targeting would be a very important technique for the genetic analysis of such microorganisms as those having no sexual generation. However, there are little research reports on the homologous or non-homologous recombination mechanism with respect to the above microorganisms, and the homologous recombination frequency of Aspergillus strains and the like is very low. Furthermore, there exist very little foreign hetero gene markers that can be used as a marker for transfomation in these strains since they originally have high drug-resistance. As a result, it has been very difficult to obtain a strain having gene-disruption or homologous recombination at a desired site except that a screening was possible based on the phenotype. Accordingly, it has been desired to develop a method for obtaining a gene-disruption strain with a high homologous recombination frequency.